U.S. patent application number 09/839752 was filed with the patent office on 2002-05-02 for therapeutic use of cis-element decoys in vivo.
Invention is credited to Dzau, Victor J., Gibbons, Gary H., Morishita, Ryuichi.
Application Number | 20020052333 09/839752 |
Document ID | / |
Family ID | 22509818 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052333 |
Kind Code |
A1 |
Dzau, Victor J. ; et
al. |
May 2, 2002 |
Therapeutic use of cis-element decoys in vivo
Abstract
The invention provides for the use of oligodeoxynucleotide
decoys for the prophylactic or therapeutic treatment of diseases
associated with the binding of endogenous transcription factors to
genes involved in cell growth, differentiation and signalling or to
viral genes. By inhibiting endogenous trans-activating factors from
binding transcription regulatory regions, the decoys modulate gene
expression and thereby regulating pathological processes including
inflammation, intimal hyperplasia, angiogenesis, neoplasia, immune
responses and viral infection. The decoys are administered in
amounts and under conditions whereby binding of the endogenous
transcription factor to the endogenous gene is effectively
competitively inhibited without significant host toxicity. The
subject compositions comprise the decoy molecules in a context
which provides for pharmacokinetics sufficient for effective
therapeutic use.
Inventors: |
Dzau, Victor J.; (Los Altos
Hills, CA) ; Gibbons, Gary H.; (Palo Alto, CA)
; Morishita, Ryuichi; (Palo Alto, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
176 FEDERAL STREET
BOSTON
MA
02110-2214
US
|
Family ID: |
22509818 |
Appl. No.: |
09/839752 |
Filed: |
April 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09839752 |
Apr 19, 2001 |
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08524206 |
Sep 8, 1995 |
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08524206 |
Sep 8, 1995 |
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08144717 |
Oct 29, 1993 |
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Current U.S.
Class: |
514/44R ;
424/450; 424/93.2 |
Current CPC
Class: |
A61K 31/70 20130101;
A61P 9/10 20180101; C12N 2310/3517 20130101; A61P 13/12 20180101;
A61P 9/14 20180101; C07H 21/00 20130101; A61K 38/00 20130101; C12N
15/113 20130101; C12N 2310/13 20130101; A61K 31/7088 20130101; A61P
17/00 20180101; A61P 37/00 20180101; A61K 48/00 20130101; A61P
35/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/44 ; 424/450;
424/93.2 |
International
Class: |
A61K 048/00; A61K
009/127 |
Claims
What is claimed is:
1. A method of modulating gene transcription in vivo within
mammalian cells, said method comprising: contacting a mammal with a
composition comprising dsDNA having a sequence specific for binding
to a transcription factor which modulates the transcription of at
least one gene, whereby said dsDNA is introduced into the nuclei of
said cells in an amount sufficient to competitively inhibit the
binding of said transcription factor to said gene, whereby the
transcription of said gene is modulated.
2. A method according to claim 1, wherein said dsDNA is capable of
episomal replication in said cell.
3. A method according to claim 1, wherein said dsDNA consists
essentially of oligonucleotides less than 100 bp in length.
4. A method according to claim 1, wherein said composition further
comprises liposomes and said dsDNA is contained within the lumen of
said liposomes.
5. A method according to claim 4, wherein said liposomes comprise
lipid and a viral coat protein.
6. A method according to claim 1, wherein said transcription factor
is E2F, AP-1 or NF.kappa.B.
7. A method according to claim 1, wherein said cells are vascular
smooth muscle cells, tumor cells or endothelial cells.
8. A method for treating a mammalian host to prevent restenosis,
said method comprising: introducing dsDNA into vascular smooth
muscle cells at the site of a vascular lesion, said cells capable
of resulting in restenosis as a result of neointima formation, in
an amount to inhibit said neointima formation, whereby said dsDNA
is characterized by having a sequence specific for binding to a
transcription factor which modulates the transcription of at least
one gene, where the transcription product of said gene is necessary
for proliferation of said cells.
9. A composition comprising a viral coat protein-liposome
containing dsDNA in the lumen of said liposome in a physiologically
acceptable medium, wherein said dsDNA is characterized by having a
sequence specific for binding to a transcription factor, wherein
said transcription product of said gene is necessary for cell
proliferation.
10. A composition according to claim 12, wherein said viral coat is
from the hemagglutinating virus Japan.
11. A composition according to claim 12, wherein said transcription
factor is E2F, AP-1 or NF.kappa.B.
12. A composition according to claim 12, wherein said dsDNA is at a
concentration in the range of about 0.1 to 20 .mu.M.
Description
TECHNICAL FIELD
[0001] The field of this invention is therapeutic treatment of
disease with double stranded nucleic acids which bind transcription
factors.
BACKGROUND
[0002] A wide variety of diseases result from the over-or
under-expression of one or more genes. Given cells may make
insufficient amounts of a protein (e.g. insulin) or too much of a
protein, be it a normal protein (e.g. TNF), a mutant protein (e.g.
an oncogene), or a non-host protein (e.g. HIV tat). The ultimate
goal of therapeutic intervention in such diseases is a selective
modulation of gene expression.
[0003] A variety of methods of transcriptional modulation in vitro
have been reported including the use of anti-sense nucleic acids
capable of binding nascent message, intracellular immunization with
dominant negative mutants.
[0004] With the broad potential therapeutic applications, massive
efforts have been extended by prominent laboratories and clinics
around the world to extend these methods in vivo. To date, the
transcription factor decoy strategy has never been successfully
adopted in vivo.
[0005] Relevant Literature
[0006] Description of the roles of transcription factors may be
found in Nevins, Science 258, 424-429 (1992); Dalton, EMBO J. 11,
11797 (1992); Yee et al. ibid. 6, 2061 (1987), Weintraub et al.,
Nature 358, 259-261 (1992), Pagano et al., Science 255, 1144-1147
(1992). Viral coat protein-liposome mediated transfection is
described by Kaneda et al., Science 243, 375 (1989). Ritzenthaler
et al. (1991) Biochem J. 280, 157-162; Ritzenthaler et al (1993) J.
Biol Chem 268, 13625-13631; Bielinska et al., Science 16, 997-1000
(1990) and Sullenger et al., Cell 63, 601-608 (1990) describe
inhibition of transcription with double stranded nucleic acids.
[0007] A general discussion concerning the mechanism of restenosis
may be found in Libby et al., Circulation 86, III-47 (1992) and
Clowes et al., J. Cardiovasc. Pharmacol. 14, S12-15 (1989).
SUMMARY OF THE INVENTION
[0008] The invention provides for the therapeutic treatment of
diseases associated with the binding of endogenous transcription
factors to genes involved in cell growth, differentiation and
signalling or to viral genes. Methods and compositions are provided
for blocking the capacity of endogenous trans-activating factors to
modulate gene expression and thereby regulating pathological
processes including inflammation, intimal hyperplasia,
angiogenesis, neoplasia, immune responses and viral infection.
[0009] The methods comprise administering to a patient double
stranded nucleic acid "decoys" in a form such that the decoys are
capable of entering target cells of the patient and specifically
binding an endogenous transcription factor, thereby competitively
inhibiting the transcription factor from binding to an endogenous
gene. The decoys are administered in amounts and under conditions
whereby binding of the endogenous transcription factor to the
endogenous gene is effectively competitively inhibited without
significant host toxicity. Depending on the transcription factor,
the methods can effect up- or down-regulation of gene expression.
The subject compositions comprise the decoy molecules in a context
which provides for pharmacokinetics sufficient for effective
therapeutic use.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1. Effect of NRE "decoy" on renin gene expression in
cultured SMG cells. SCA-9 cells expressed renin as shown by
immunohistochemistry (panel A). Primer extension analysis
demonstrated that this was exclusively Ren 2 (panel B). These cells
effectively took up FITC labeled double stranded decoy oligomer
corresponding to the NRE (panel C). RNA was prepared 24 hours after
transfection from control and "decoy" treated SCA-9 cells. Note
that Ren 1 mRNA could be observed after exposure of the cells to
the NRE decoy (panel B, lane 4).
[0011] FIG. 2. Detection of the NRE binding protein in cultured SMG
cells. Nuclear extracts were prepared from SCA-9 cells and
incubated with 32P end-labeled mouse renin NRE oligonucleotide.
Competition analysis was performed with 50- and 100-fold excess of
unlabeled NRE oligonucleotide. Note that a specific NRE:NRE binding
protein complex formation was observed.
[0012] FIG. 3. In Vivo Expression of CAT in the Mouse SMG. Ten ug
of renin gene CAT construct was transfected directly into the SMG
of DBA/2J mice using the HVJ-DNA-Liposome technique. Four days
after transfection, the SMG was removed, cell extracts prepared and
CAT activity measured.
[0013] FIG. 4. Schematic diagram of the factors influencing renin
gene expression. The hatched bar represents the CRE/NRE region
present in the renin gene. The CRE binding protein and the NRE
binding protein compete for binding to this region. The triangle
represents the 150 base pair insert which is present in the Ren 2
gene.
[0014] Ren 1 expression: in the kidney, the CRE binding protein
binds tighter, blocking the binding of the NRE binding protein, and
allowing expression of the Ren 1 gene. In the SMG, an inhibitory
protein forms an inactive complex with the CRE binding protein,
allowing the NRE binding protein to bind, silencing expression of
Ren 1.
[0015] Ren 2 expression: The 150 bp insertion interferes with the
NRE function in the Ren 2 gene, resulting in Ren 2 expression in
the SMG and kidney.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0016] Methods and compositions are provided for modulating gene
expression in vivo. The methods involve administering a composition
to a patient so as to introduce into a target cell molecular decoys
comprising double-stranded nucleic acid, usually DNA, to which
transcription factors endogenous to the target cell bind. Various
methods are employed for in vivo administration of the decoys such
that sufficient decoys enter into the target cells to competitively
inhibit transcription factor binding to an endogenous gene
regulatory region.
[0017] The compositions which are employed comprise "decoys":
double-stranded nucleic acid molecules with high binding affinity
for the targeted transcription factors. The targeted transcription
factors are endogenous, sequence-specific double-stranded DNA
binding proteins which modulate (increase or decrease) the rate of
transcription of one or more specific genes in the target cell.
Essentially any such transcription factor (henceforth,
"transcription factor") can be targeted so long as a specific decoy
capable of competitively inhibiting binding to the endogenous gene
can be identified. Numerous transcription factors and their binding
sequences are known in the art as are methods for identifying such
complements, see e.g. Wang and Reed (1993) Nature 364, 121 and
Wilson et al. (1991) Science 252, 1296. As used herein, endogenous
means that the gene or transcription factor is present in the
target cell at the time the decoy is introduced.
[0018] The transcription factors will, for the most part and
depending on the clinical indication, regulate the transcription of
genes associated with cell growth, differentiation and signalling
or viral genes resident in the target cell. Examples include genes
necessary for mitosis, particularly going from G.sub.o to S, such
as proteins associated with check points in the proliferative
cycle, cyclins, cyclin dependent kinases, proteins associated with
complexes, where the cyclin or cdk is part of the complex,
Rosenblatt et al., Proc. Natl. Acad. Sci. 89, 2824 (1992) and
Pagano et al., Science 255, 1144 (1992). Often such genes or the
transcription factors themselves will be oncogene products or
cellular counterparts, e.g. fos, jun, myc, etc. Other examples
include genes encoding secreted proteins and peptides such as
hormones e.g. growth factors, cytokines, e.g. interleukins,
clotting factors, etc. Target transcription factors also include
host and host-cell resident viral transcription factors which
activate viral genes present in infected host cells.
[0019] Preferred target transcription factors are activated (i.e.
made available in a form capable of binding DNA) in a limited
number of specifically activated cells. For example, a stimulus
such as a wound, allergen, infection, etc may activate a metabolic
pathway that is triggered by the transient availability of one or
more transcription factors. Such transcription factors may be made
available by a variety of mechanisms such as release from
sequestering agents or inhibitors (e.g. NF.kappa.B bound to IkB),
activation by enzymes such as kinases, translation of sequestered
message, etc. Desirably, the target transcription factor(s) will be
associated with genes other than genes whose lack of expression
results in cytotoxicity. For the most part, it is desirable not to
kill the cell, but rather to inhibit or activate specific gene
transcription.
[0020] Exemplary transcription factors and related cis elements,
the cellular processes impacted and therapeutic indication
include:
1 Cis-elemnt Therapeutic Txn Factor Cellular Process Application
E2F cell proliferation neointimal hyper- plasia, neoplasia
glomerulonephritis, angiogenesis, inflammation AP-1 cell growth,
differentiation, neointimal hyper- growth factor expression plasia,
cardiac myocyte growth/ differentiation NFkB cytokine expression,
leukocyte inflammation, immune adhesion molecule expression,
response, transplant oxidant stress response, cAMP rejection,
ischemia- and protein kinase C activation, reperfusion injury, Ig
expression glomerulonephritis SSRE response to shear stress: growth
neointimal hyper- factor expression vasoactive plasia, bypass
grafts, substances, matrix proteins, angiogenesis, adhesion
molecules. collateral formation. CREB cAMP response cAMP activated
events MEF-2 cardiac myocyte differentiation cardiac myocyte and
hypertrophy differentiation and growth. CArG box cardiac myocyte
differentiation cardiac myocyte growth and differ- entiation. tax
viral replication HTLV infection VP16 viral replication Herpes
infection TAR/tat viral replication HIV infection GRE/HRE
glucocorticoid, mineralocorticoid steroid hormone MRE induced
events processes e.g. (breast or prostate cell growth). Heat shock
heat shock response cellular stresses e.g. RE ischemia, hypoxia SRE
growth factor responses cell proliferation/ differentiation AP-2
cAMP and protein kinase cell proliferation. response, retinoic acid
response sterol modulation of LDL cholesterol hypercholesterolemia
response receptor expression element TRE Transforming growth factor
beta cell growth, differ- TGFb induced cellular processes
entiation, migration, responsive angiogenesis, intimal element
hyperplasia, matrix generation, apoptosis.
[0021] The length, structure and nucleotide sequence of the decoy
will vary depending on the targeted transcription factor, the
indication, route of administration, etc. For example, targeted
transcription factors frequently bind with different degrees of
affinity to a variety of sequences, normally sharing a high degree
of homology. Accordingly, one may choose to use a sequence
associated with a particular target gene or use a consensus
sequence based on the nucleotide at each site which occurs most
frequently in the binding sequences to which the particular
transcription factor binds. For example, when targeting a host
transcription factor involved in viral transcription, it may be
possible to minimize undesirable effects on host transcriptions
preferable by employing the viral-specific binding sequence.
Similarly, where transcription is mediated by a multimeric complex,
it is often desirable to target a single transcription factor to
minimize effects on non-targeted genes. For example, in the case of
Herpes virus transcription, one may target the viral VP16 without
concomitant targeting of the promiscuous host Oct protein.
[0022] In addition to binding affinity, the decoys are also
selected for binding specificity. Desirably, the decoys will be
highly specific for the target transcription factor(s) such that
their effect on nontarget cells and nontargeted metabolic processes
of target cells are minimized. Such selection is accomplished in
vitro by comparative binding to known transcription factors and
nuclear extracts and in culture and in vivo by assaying nontargeted
cell function and effects on nontargeted cell types.
[0023] The decoys contain sufficient nucleotide sequence to ensure
target transcription factor binding specificity and affinity
sufficient for therapeutic effectiveness. For the most part, the
target transcription factors will require at least six base pairs,
usually at least about eight base pairs for sufficient binding
specificity and affinity. Frequently, providing the decoys with
flanking sequences (ranging from about 5 to 50 bp) beside the
binding site enhance binding affinity and/or specificity.
Accordingly, cis element flanking regions may be present and
concatemer oligonucleotides may be constructed with serial
repetitions of the binding and/or cis element flanking
sequences.
[0024] In one embodiment, the decoys are non-replicative
oligonucleotides fewer than 100 bp, usually fewer than 50 bp and
usually free of coding sequence, being primarily from the
non-coding 5' region of a gene. Alternatively, the decoys may
comprise a portion of a larger plasmid, including viral vectors,
capable of episomal maintenance or constitutive replication in the
target cell to provide longer term or enhanced intracellular
exposure to the decoy sequence. Plasmids are selected based on
compatibility with the target cell, size and restriction sites,
replicative frequency, copy number maintenance, etc. For example,
plasmids with relatively short half-lives in the target cell are
preferred in situations where it is desirable to maintain
therapeutic transcriptional modulation for less than the lifetime
of the target cell. Exemplary plasmids include pUC expression
vectors driven by a beta-actin promoter and CMV enhancer, vectors
containing elements derived from RSV or SV40 enhancers, etc. The
adeno-associated viral vector preferentially integrates in
chromosome 19 and may be utilized for longer term expression.
[0025] The oligonucleotides which are employed may be naturally
occurring or other than naturally occurring, where the synthetic
nucleotides may be modified in a wide variety of ways, see e.g.
Bielinska et al (1990) Science 250, 997. Thus, oxygens may be
substituted with nitrogen, sulfur or carbon; phosphorus substituted
with carbon; deoxyribose substituted with other sugars, or
individual bases substituted with an unnatural base. In each case,
any change will be evaluated as to the effect of the modification
on the binding of the oligonucleotide to the target transcription
factor, as well as any deleterious physiological effects. These
modifications have found wide application for "anti-sense"
oligonucleotides, so that their safety and retention of binding
affinity are well established in the literature. See, for example,
Wagner et al., Science 260, 1510-1513 (1993). The strands may be
synthesized in accordance with conventional ways using
phosphoramidite synthesis, commercially available automatic
synthesizes, and the like.
[0026] The administered compositions may comprise individual or
mixtures of decoys. Usually the mixture will not exceed 4 different
decoys usually not exceed 2. The decoys are administered to a host
in a form permitting cellular internalization of the decoys in an
amount sufficient to competitively inhibit the binding of the
targeted transcription factor to an endogenous gene. The host is
typically a mammal, usually a human. The selected method of
administration depends principally upon the target cell, the nature
of the decoy, the host, the size of the decoy. Exemplary methods
are described in the examples below; additional methods including
transfection with a retrovirus, viral coat protein-liposome
mediated transfection, lipofectin etc. are described in Dzau et
al., Trends in Biotech 11, 205-210 (1993).
[0027] Where administered in liposomes, the decoy concentration in
the lumen will generally be in the range of about 0.1 uM to 50 uM
per decoy, more usually about 1 uM to 10 uM, most usually about 3
uM. For other techniques, usually one will determine the
application rate empirically, using conventional techniques to
determine desired ranges.
[0028] In some situations it may be desirable to provide the decoy
source with an agent which targets the target cells, such as an
antibody specific for a surface membrane protein on the target
cell, a ligand for a receptor on the target cell, etc. For example,
for intervention in HIV infection, cells expressing HIV gene
products or CD4 may be specifically targeted with gene product or
CD4-specific binding compounds. Also, where liposomes are involved,
one may wish to include proteins associated with endocytosis, where
the proteins bind to a surface membrane protein associated with
endocytosis. Thus, one may use capsid proteins or fragments thereof
tropic for a particular cell type, antibodies for proteins which
undergo internalization in cycling, proteins that target
intracellular localization and enhance intracellular half-life.
[0029] The application of the subject therapeutics are preferably
local, so as to be restricted to a histological site of interest
e.g. localized inflammation, neoplasia or infection. Various
techniques can be used for providing the subject compositions at
the site of interest, such as injection, use of catheters, trocars,
projectiles, pluronic gel, stents, sustained drug release polymers
or other device which provides for internal access, or the like.
Where an organ or tissue is accessible because of removal from the
patient, such organ or tissue may be bathed in a medium containing
the subject compositions, the subject compositions may be painted
onto the organ, or may be applied in any convenient way.
Alternatively, systemic administration of the decoy using e.g.
lipofection, liposomes with tissue targeting (e.g. antibody), etc.
may be practiced. Systemic administration is most applicable where
the distribution of the targeted transcription factor is primarily
limited to targeted cell types, e.g. virus-specific transcription
factors limited to infected cells, mutant oncogenic transcription
factors limited to transformed cells, etc.
[0030] Optimal treatment parameters will vary with the indication,
decoy, clinical status, etc., and are generally determined
empirically, using the guidance provided herein. Several exemplary
indications, routes and vehicles of administration and decoy
combinations are disclosed in the following table.
2 PLASMD/ INDICATION ROUTE VEHICLE OLIGO HIV infection intravenous
gp160 in TAR con- inj. neutral liposomes taining oligo solid tumor
intratumoral tumor-specific Ab E2F inj. with liposomes Inflammatory
skin topical polymer NF.kappa.B, E2F disease and dermatitis
Hypercholesterol- intravenous cationic liposomes sterol re- emia
inj. portal asialoglycoprotein sponsive vein inj. receptor
targeting element to with lipsomes increase LDL receptors vein
bypass grafts topical/ polymer, E2F intralumina liposomes 1
glomerulonephritis intravenous, polymer, E2F, NF.kappa.B intrarenal
liposomes myocardial intracoronary liposomes, NF.kappa.B, E2F,
infarction polymer AP-1 organ transplant intra- liposomes,
NF.kappa.B esp. cardiac/renal vascular, ex polymer vivo
[0031] A wide variety of indications may be treated, either
prophylactically or therapeutically with the subject compositions.
For example, prophylactic treatment may inhibit mitosis or
proliferation or inflammatory reaction prior to a stimulus which
would otherwise activate proliferation or inflammatory response,
where the extent of proliferation and cellular migration may be
undesirable. Similarly, a therapeutic application is provided by a
situation where proliferation or the inflammatory response is about
to be initiated or has already been initiated and is to be
controlled. The methods and compositions find use, particularly in
acute situations, where the number of administrations and time for
administration is relatively limited.
[0032] Conditions for treatment include such conditions as
neoproliferative diseases including inflammatory disease states,
where endothelial cells, inflammatory cells, glomerular cells may
be involved, restenosis, where vascular smooth muscle cells are
involved, myocardial infarction, where heart muscle cells may be
involved, glomerular nephritis, where kidney cells are involved,
hypersensitivity such as transplant rejection where hematopoietic
cells may be involved, cell activation resulting in enhancement of
expression of adhesion molecules where leukocytes are recruited, or
the like. By administering the decoys to the organ ex vivo prior to
implantation and/or after implantation, upregulation of the
adhesion molecules may be inhibited. Adhesion molecules include
homing receptors, addressing, integrins, selecting, and the
like.
[0033] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1.
Transfection of E2F Decoys into Cultured Cells.
[0034] For the nuclear extracts, vascular smooth muscle cells
("VSMCs") were stimulated by serum until confluent. After
confluent, the cells were made quiescent by placing in serum free
medium for 48 h. After the transfection of decoy
oligodeoxynucleotides ("ODN"; 14 bp) essentially as described in
Morishita et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 8474-8478,
cells were stimulated by 5% serum. After 6 h, RNA was extracted by
RNAzol (Tel-Test Inc, Texas) Chomczynski and Sacchi (1987) Anal
Biochem 162, 156-159. Levels of PCNA, cdc2 and beta-actin mRNAs
were measured by RT-PCR (Morishita et al. (1993) supra). The PCNA
primer (nucleotides 150-177 of rat PCNA cDNA) and the cdc2 5'
primer (nucleotides 54-75 of human cdc2 CDNA) were previously
described (Morishita et al. (1993) supra). The primers
complementary to the rat beta-actin gene were obtained from
Clontech Laboratories Inc. (Palo Alto, Calif.). Aliquots of RNA
were amplified simultaneously by PCR (30 cycles) and compared with
a negative control (primers without RNA). Amplification products
were electrophoresed through 2 % agarose gels stained with ethidium
bromide. A gel mobility shift assay was performed as previously
described (Horiuchi et al., J. Biol. Chem. 266, 16247-16254
(1991).
[0035] The 14 bp double-strand ODN effectively abolished the
binding of the E2F transcription factor to a specific binding site
in serum stimulated VSMCs. See also, Hiebert et al. (1989) PNAS 86,
3594. Transfection of the E2F decoy ODN markedly inhibited the
induction of c-myc, cdc2 and PCNA mRNA expression in response to
serum stimulation. The E2F decoy ODN had no effect on beta-actin
mRNA expression. Furthermore, the control missense E2F element ODN
containing two base pair substitutions that eliminate E2F binding
failed to inhibit the induction of c-myc, cdc2 and PCNA RNA
expression in response to serum stimulation. In association with
effective inhibition of cell cycle regulatory gene expression,
transfection of the 14 bp E2F decoy also abolished serum-stimulated
VSMC proliferation. In contrast, the missense E2F element ODN had
no effect on mitogenesis induced by serum.
[0036] To further confirm the specificity of this response to the
E2F decoy, a 30 bp double-stranded ODN which contained two 8 base
pair E2F cis elements capable of specific binding to E2F was
employed (Weintraub et al., Nature 358, 259-261 (1992)). In the 30
bp E2F decoy the fifth nucleotide of the 8 bp E2F cis elements was
changed from C to G. Despite these differences in flanking
sequences and nucleotide composition, both E2F decoys effectively
bind E2F and inhibit serum-stimulated vascular smooth muscle cell
(VSMC) proliferation. Moreover, a 30 bp missense E2F element ODN
with 5 bp substitutions with the 8 bp E2F consensus elements fails
to bind E2F and also fails to inhibit serum-stimulated VSMC
proliferation. Thus, these in vitro studies documented that
transfection of the E2F cis element decoy ODN binds the E2F
transcription factor, blocked the induction of cell cycle
regulatory gene expression and inhibited VSMC proliferation in a
sequence specific manner.
Example 2.
E2F Decoys In Vivo.
[0037] Liposomes were prepared as follows: Phosphatidylserine
phosphatidylcholine, and cholesterol were mixed in a weight ratio
(1:4.8:2) to create a lipid mixture. Lipid was hydrated in a
balanced salt solution containing ODN (110 nmol). Purified HVJ(Z)
strain was inactivated by UV radiation just before use. The
liposome suspension was mixed with HVJ (Z strain) (20,000
hemagglutinating units), incubated, then free HVJ removed by
sucrose density gradient centrifugation. The final concentration of
encapsulated DNA was calculated as previously reported (Morishita
et al. (1993) supra). This method results in a more rapid cellular
uptake and nuclear concentration, leading to a 100-fold higher
transfection efficiency of ODN than lipofection or passive uptake
methods.
[0038] The sequences of the phosphorothioate ODN utilized:
3 decoys-1 5'-CTAGATTTCCCGCG-3' 3'-TAAAGGGCGCCTAG-5' mismatched-1
5'-CTAGATTTCGAGCG-3' 3'-TAAAGCTCGCCTAG-5'
[0039] We also examined another set of decoy ODNs containing two
binding sites:
4 decoys-2: 5'-GATCAAAAGCGCGAATCAAAAGCGCGAATC-3'
3'-CTAGTTTTCGCGCTTAGTTTTCGCGCTTAG-5' mismatched-1;
5'-GATCAAAGAACTGAATCAAAGAACTGAATC-3'
3'-CTAGTTTCTTGACTTAGTTTCTTGACTTAG-5'
[0040] Rat aortic VSMC (passage 4-10) were studied in a confluent,
quiescent state in serum-free media (Morishita et al, J. Clin.
Invest. 91, 2580-2585 (1993)). The cells were incubated with
hemagglutinating virus Japan (HVJ) liposomes (3 .mu.M) at 4.degree.
C. for 5 min and 37.degree. C. for 30 min. Three days after
transfection in either calf serum (CS) or serum-free media, cell
number was determined by Coulter-Counter (Coulter, Fla.).
Example 3.
Effect of Decoy ODN on in Vivo Gene Expression.
[0041] A 2 French Fogarty catheter was used to induce vascular
injury in male Sprague-Dawley rats (400-500 g; Charles River
Breeding Laboratories) (Hanke et al., Circ. Res. 67, 651-659
(1990)). These rats were anesthetized, and a cannula introduced
into the left common carotid via the external carotid artery. After
vascular injury of the common carotid, the distal injured segment
was transiently isolated by temporary ligatures. The HVJ complex
was infused into the segment and incubated for 10 min at room
temperature. No adverse neurological or vascular effects were
observed in any animal undergoing this procedure.
[0042] For RNA analysis, vessels were harvested at 6 h, (c-myc and
beta-actin) and one day (cdc2 kinase, PCNA and beta-actin) after
transfection. RNA was extracted from mismatched or E2F decoy ODN (3
.mu.M) treated injured or untreated intact vessels by RNAzol
(Tel-Test Inc., TX). RT-PCR was performed as described above. For
BrdU staining, BrdU was injected into rats (100 mg/kg subcutaneous
and 30 mg/kg intraperitoneal at 18 h prior, and then 30 mg/kg
intraperitoneal at 12 h prior to sacrifice (Hanke et al., supra)).
Rats were sacrificed after day four after transfection. The carotid
artery was removed after perfusion-fixation with 4%
paraformaldehyde, and processed for immunohistochemistry in a
standard manner using anti-BrdU antibodies (Amersham). The
proportion of BrdU positive cells was determined by cell counts
under light microscopy in a blinded fashion.
[0043] Transfection procedures were described above. HVJ-ODN
complex (3 .mu.M) was administered into the rat carotid injured
arteries. At two weeks after transfection, rats were sacrificed and
vessels were perfusion-fixed with 4% paraformaldehyde. Three
individual sections from the middle of transfected segments were
analyzed. In addition, three sections from the middle section of
the injured untransfected region were also analyzed. Animals were
coded so that the operation and analysis were performed without
knowledge of which treatment individual animals received. Intimal
and medial areas were measured on a digitizing tablet (Southern
Micro Instruments, GA). Analysis of variance with subsequent
Duncan's test was used to determine significant differences in
multiple comparisons. P<0.05 was considered significant.
[0044] The scrambled and progesterone responsive element (PRE)
decoy sequences utilized as control ODNs are as follows:
5 Scrambled 5'-TCCAGCTTCGTAGC-3' decoys: 3'-GAAGGATCGATCG-5' PRE:
5'-GATCCTGTACAGGATGTTCTAGCT- ACA-3'
3'-CTAGGACATGTCCTACAAGATCGATGT-5'
[0045] At one day after balloon injury mRNA levels of c-myc, cdc2
and PCNA were elevated in carotid vessels transfected with the
control missense E2F element ODN as detected by RT-PCR. However, in
vivo transfection of the E2F cis element decoy ODN resulted in a
marked decrease in c-myc, cdc2 and PCNA mRNA levels to barely
detectable levels similar to uninjured vessels. Moreover, the E2F
cis element decoys significantly inhibited bromodeoxyuridine (BrdU)
incorporation (a marker of DNA synthesis) within the vessel wall at
4 days post-injury as compared to the missense E2F ODN material.
Furthermore, transfection of the E2F decoy ODN (n=8) resulted in a
marked suppression of neointima formation at two weeks after
angioplasty compared to vessels treated with the HVJ-liposome
complex alone (n=5) or mismatched control decoy ODN-treated vessels
(n=8). The selectivity of the decoy ODN effect was further
confirmed by the fact that the inhibition of the neointima
formation was limited to the area of intraluminal transfection
(neointima/medial ratio; transfected lesions=0.291.+-.0.061 vs.
untransfected lesions=1.117.+-.0.138, P<0.01). In contrast, the
adjacent injured carotid segments outside the area of decoy
transfection exhibited neointimal lesions similar to the mismatched
ODN-treated control. Moreover, neither transfection of scrambled
decoys (14 bp) nor the progesterone response element decoys (27 bp)
(Klein-Hitpass et al., Cell 60, 247-257 (1990)) resulted in the
inhibition of neointima formation.
[0046] The specificity of the inhibitory effect of the decoy ODN
against E2F on neointima information is supported by several lines
of evidence: (1) the two different E2F decoy ODN bind to the E2F
transcription factor in a sequence specific manner and transfection
of the E2F decoy ODN completely inhibited VSMC proliferation in
vitro, whereas the mismatched ODN did not; (2) the in vitro and in
vivo experiments documented that the E2F decoy ODN selectively
inhibited the expression of the targeted cell cycle regulatory
genes (c-myc, PCNA and cdc2 kinase) transactivated by E2F, but not
beta-actin (presumably, other E2F transactivated genes such as
c-myb might be inhibited); (3) the E2F decoy ODN specifically
reduced a quantitative marker of cell cycle progression in vivo
(BrdU labeling); (4) the administration of the E2F decoy, but not
mismatched, ODN markedly inhibited neointima formation; (5) the
prevention of neointima information was limited to the area
transfected with the decoy ODN; and (6) neither scrambled nor PRE
cis element decoys inhibited the neointima formation.
Example 4.
Expression of Renin in Cultured Submanibular Gland (SMG) Cell
Line
[0047] To evaluate the molecular mechanisms which regulate tissue
specific renin gene expression, we employed a cell line (SCA-9)
derived from a SMG tumor. This line, derived from a two renin gene
mouse (Swiss Webster) has been reported to contain renin. Using
immunohistochemistry and primer extension analysis, we confirmed
that this cell line expresses renin, as shown by
immunohistochemistry (FIG. 1A). Moreover, consistent with the SMG
origin of this cell line, only the Ren2 gene is transcribed (FIG.
1B). To elucidate the mechanism behind the silencing of the Ren1
gene in this cell line, we examined if the negative response
element, NRE, binding protein was present in these cells. Nuclear
extracts were prepared from cultured SMG cells and gel mobility
shift assay was performed using .sup.32P-NRE oligonucleotide as a
probe (FIG. 2). A specific protein: DNA complex was observed which
could be specifically competed with unlabeled NRE
oligonucleotide.
[0048] These results show that the pattern of renin expression in
this cell line is similar to that seen in the SMG in vivo;
validating the use of these cells for studies of the mechanism by
which renin expression is regulated.
Example 5.
Use of "Decoy" to Validate the Importance of Cis-Trans
Interactions
[0049] We examined the feasibility of the "decoy" approach to test
whether the NRE:NRE binding protein interaction is responsible for
the silencing of the Ren1 gene in the SMG and in the cell line
derived from the SMG. This assay is based on the in vivo
competition for trans acting factors. The competition is between
the endogenous cis elements found in the target gene and
exogenously added oligonucleotides corresponding to that cis
sequence. This competition will attenuate the authentic cis-trans
interaction, result in the removal of the trans factors from the
cis element, with the subsequent modulation of gene expression.
[0050] An FITC labeled-double stranded oligonucleotide (33 mer),
corresponding to the NRE were introduced into the SMG cell line
using the HVJ-liposome technology. The cells were incubated with
HVJ-liposome containing NRE decoy at 4.degree. C. for 10 minutes
and then at 37.degree. C. for 30 minutes. The cells readily took up
the oligomers as evidenced by the intense fluorescent signal which
was localized in the nucleus (FIG. 1C). The cells were harvested 24
hours later, RNA was prepared and subjected to the primer extension
analysis (FIG. 1B) to examine the expression of the Ren1 and Ren2
genes. Consistent with our hypothesis, introduction of the NRE
sequences into these cells resulted in the induction of Ren1 gene
expression. The expression of the Ren2 gene was not influenced by
the administration of the NRE oligonucleotide.
[0051] These results show that NRE:NRE binding protein interaction
is responsible for the silencing of Ren1 expression in this cell
line and demonstrate the feasibility of using the "decoy" approach
to examine the cis-trans interactions responsible for the
regulation of renin gene expression.
Example 6.
In Vivo Gene Transfer into the SMG
[0052] We transfected 10 ug of our expression vectors into the SMG
of a DBA/2J mouse using the HVJ-Liposome-DNA complex as describe
above. This was accomplished by injecting directly into multiple
regions of the gland with a total volume of 100 ul using a 27 gauge
needle. Three days after transfection, the SMG was removed,
homogenized and the supernatant assayed for CAT activity (FIG. 3).
Transfection of pUtk-CAT directed the expression of CAT (Horiuchi
et al. (1991) supra). The presence of the Ren1 Xba fragment
(containing the CRE/NRE region) decreased CAT expression by
approximately 90%. Deletion of the NRE sequence by in vitro
mutagenesis resulted in the recovery of CAT expression. These
results demonstrate that it is possible to use in vivo gene
transfer to examine the expression of genes in the SMG in vivo.
[0053] It is evident from the above results, that the subject
methods and compositions provide opportunities for preventing
injury from acute proliferative diseases or diseases involving
expression of proteins, such as proteins associated with cell
division, adhesion and/or migration. Thus, the subject methods and
compositions can provide safe prophylactic and therapeutic
treatments for a number of diseases associated with cellular
proliferation and inflammation, resulting in pathogenic
conditions.
[0054] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0055] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *